Summary While our comprehension of heart development has changed and gained great insight in recent years, there is still a fundamental lack of understanding with respect to the establishment of atrial/ventricular specification. This gap in knowledge hinders our ability to fully comprehend the morphogenetic events that lead to normal development, and how errors in this process can lead to congenital heart defects. As these make up the most common birth defects, impacting ~1% of newborns, a better comprehension of early heart development may instruct us to better model congenital heart defects in the lab and ultimately improve the lives of a great number of people. This proposal aims to explore the mechanisms during early embryogenesis that ensure correct formation of the four-chambered heart. Specifically, I aim to elucidate the underlying signaling pathways that specify ventricular cardiovascular cells during development. I will utilize lineage tracing techniques to label and monitor the ventricular precursors over time, which will allow me to identify key players at every stage leading up to the formation of the ventricular chambers. I will isolate and characterize prospective ventricular cells at the cardiac mesoderm stage, the cardiac crescent stage, and the early heart tube stage. These data will serve to identify novel pathways and to establish a temporally defined network at play during ventricular cardiac development. Furthermore, my initial results from these experiments have provided evidence that the Notch signaling pathway may be important for early ventricular progenitor patterning and differentiation. I will explore this finding further using ex vivo embryo culture and the human embryonic stem cell system to modulate Notch pathway activity during cardiomyocyte differentiation. These data will help us to understand early how early signaling events affect atrial-ventricular lineage segregation. The research laid out in this proposal effectively combines complementary model systems and previously unavailable tools and concepts to examine the specification of ventricular cells during heart development in an innovative new approach. Acquiring an in-depth understanding of ventricular cell development is of high significance because it holds the promise of uncovering novel disease-relevant mechanisms and developmental concepts. In addition, it is well established that applying developmental principles can significantly improve pluripotent stem cell differentiation strategies. The insights we will obtain from our systematic approach can thus be translated to generate defined cardiac cell types in vitro, which are expected to harbor broad clinical and translational potential for cardiovascular disease.